Five-helix protein

ABSTRACT

Five-Helix protein, which comprises the three N-helices and at least two, but not three, of the three C-helices of the trimer-of-hairpin structure of HIV gp41, separated by linkers, such as amino acid residue linkers, is disclosed. Six-Helix protein, which includes the three N-helices and the three C-helices of the trimer-of-hairpin structure of HIV gp41, separated by linkers, is also disclosed.

RELATED APPLICATION(S)

This application is a divisional of U.S. application Ser. No. 11/151,598filed Jun. 13, 2005, which issued as U.S. Pat. No. 7,504,224, which is acontinuation of U.S. application Ser. No. 09/738,945, filed Dec. 15,2000, which issued as U.S. Pat. No. 7,053,179 and which claims thebenefit of U.S. Provisional Application No. 60/171,042, filed Dec. 16,1999 and U.S. Provisional Application No. 60/234,572, filed Sep. 22,2000. The entire teachings of the above applications are incorporatedherein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by Grant Number PO1 GM56552 from National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

HIV is the virus that is responsible for the worldwide AIDS epidemic.The initial stages of HIV infection involve the fusion of the viralmembrane with the target cell membrane, a process that injects the viralcontents into the cellular cytoplasm. On the viral side, the molecularcomplex responsible for the fusion activity contains the surface proteingp120 and the transmembrane protein gp41. It is currently believed thatgp120 interacts with the proteins CD4 and coreceptors on the targetcell, resulting in a conformational change that causes gp41 to insertits amino terminus (fusion peptide region) into the target cellmembrane. This structural rearrangement promotes the fusion of virus andcellular membranes through a poorly understood mechanism.

SUMMARY OF THE INVENTION

The present invention relates to a novel protein, referred to as Five(5)-Helix or Five-Helix protein, that, under the conditions describedherein, folds into a stable structure, binds a peptide (referred to asC34) that corresponds to the C-peptide region of HIV gp41 or a portionof the region, and inhibits HIV infection of mammalian cells, such ashuman cells. Five-Helix is made up of the three N-helices and at leasttwo, but not three, of the three C-helices of the trimer of hairpinstructure of HIV gp41, separated by linkers, such as amino acid residuelinkers. That is, Five-Helix includes the three N-helices and at leasttwo of the three C-helices of HIV gp41. It can also include a portion ofthe third C-helix, but does not include the entire third C-helix. Ineach case, the helices are separated by linkers, preferably amino acidresidue linkers, between the preceding and following helices. In oneembodiment, Five-Helix can be represented as:N-linker-C-linker-N-linker-C-linker-N, wherein N represents an N-helixand C represents a C-helix or C-helix portion. As used herein, the termFive-Helix or Five-Helix protein encompasses all such embodiments (thoseincluding three N-helices and two or more, but less than three completeC-helices, separated by appropriate linkers). The amino acid compositionof Five-Helix can vary greatly, provided that Five-Helix presents asurface that is structurally complementary to the C-peptide region ofHIV gp41 protein and, preferably, binds C34 or the C-peptide region ofgp41, as peptides or part of gp41 as a whole. That is, the remaining(interacting) surface of Five-Helix (the C-peptide binding site, all ora portion of which is not occupied by a C-peptide) must be presented insuch a manner (conformation) that it is available to bind the C-peptideregion of HIV gp41. In the case of vaccine and therapeutic applicationsof Five-Helix, Five-Helix must bind (be capable of binding) C34 or theC-peptide region of HIV gp41. In the cases in which Five-Helix is usedas a drug-screening tool or an antibody-screening tool, Five-Helix neednot bind (need not be capable of binding) C34 or the C peptide region ofHIV gp41.

In one embodiment, Five-Helix has the amino acid sequence of SEQ IDNO.: 1. In other embodiments, Five-Helix presents a surface that isstructurally complementary to the C-peptide region, preferably binds C34or the C-peptide region and has an amino acid sequence that differs fromthat of SEQ ID NO.: 1 by addition, deletion, substitution or alterationof at least one amino acid residue. The order of the N-helices andC-helices of Five-Helix can also vary, provided that the conformation issuch that the exposed protein presents a surface structurallycomplementary to the C-peptide region of HIV gp41. The linkers can be ofany length or composition, provided that the Five-Helix proteinconformation, described above, is retained. Five-Helix can be an L-aminoacid protein, a D-amino acid protein or a combination of L-amino acidresidues and D-amino acid residues; these residues can be modifiedresidues.

The present invention further relates to DNA encoding Five-Helix;methods of producing Five-Helix; methods in which Five-Helix is used,such as in methods of inhibiting entry of HIV into mammalian cells,including human cells, and methods of eliciting an immune response in anindividual, such as a human; methods in which DNA encoding Five-Helix isused, such as in gene therapy methods; genetically engineered cells,such as bacteria, human and other mammalian and other eukaryotic cells,which contain and express Five-Helix protein-encoding DNA and methods ofusing such cells (e.g., for gene therapy or Five-Helix production);compositions, such as pharmaceutical compositions, which includeFive-Helix; Five-Helix complex comprising Five-Helix and a componentthat binds HIV envelope protein (e.g., gp120); compositions, such aspharmaceutical compositions, which include Five-Helix complex;antibodies, particularly neutralizing antibodies which bind Five-Helixand methods in which such antibodies are used, such as methods ofreducing HIV infection; and methods of identifying molecules orcompounds that inhibit HIV infection of cells and/or bind the Five-Helixprotein.

Five-Helix is useful as an anti-HIV therapeutic agent, a prophylacticagent or drug to prevent HIV infection, a reagent for identifying(screening for) or designing other anti-HIV therapeutics orprophylactics, and an immunogen to elicit antibodies that prevent orreduce HIV infection. In a specific embodiment, the invention relates toa method of identifying a compound or molecule that binds Five-Helix andinhibits HIV infection of mammalian cells, wherein the compound ormolecule to be assessed is referred to as a candidate inhibitor,comprising combining a candidate inhibitor and Five-Helix, underconditions appropriate for binding of an inhibitor and Five-Helix tooccur and determining if binding occurs, wherein if binding occurs, thecandidate inhibitor is a compound or molecule that binds Five-Helix. Themethod optionally further comprises determining if the compound ormolecule that binds Five-Helix inhibits HIV infection of mammalian(e.g., human) cells, such as in a cell-based assay. Such a compound ormolecule will inhibit (totally or partially) HIV infection of cells(e.g., by preventing or interfering with formation of thetrimer-of-hairpins).

In another embodiment, the invention relates to a method of eliciting animmune response to HIV in an individual, comprising introducing, by anappropriate route, a composition comprising Five-Helix and aphysiologically acceptable carrier, in a dose sufficient to elicit theimmune response in the individual. Vaccines comprising Five-Helix (or avariant or portion thereof) in a physiologically acceptable carrier arethe subject of this invention.

Also the subject of the present invention is Six (6)-Helix protein,which comprises three N-helices and three C-helices of HIV gp41, joinedby linkers, such as amino acid residue linkers. In one embodiment,Six-Helix protein comprises the amino acid sequence of SEQ ID NO.: 2. Inother embodiments, the amino acid sequence of Six-Helix differs fromthat of SEQ ID NO.: 2 by addition, deletion, substitution or alterationof at least one amino acid residue. Six-Helix protein is useful not onlyfor producing Five-Helix, but also as a negative control in screeningfor drugs that inhibit membrane fusion.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing in color. Copiesof this patent with color drawing(s) will be provided by the Patent andTrademark Office upon request and payment of the necessary fee.

FIGS. 1A and 1B illustrate targeting HIV-1 membrane fusion. FIG. 1A is aschematic of HIV-1 membrane fusion depicting events that promoteformation of the gp41 trimer-of-hairpins. The N-terminal fusion peptideof gp41 (red), inaccessible in the native state, inserts into targetcell membranes following gp120 interaction with CD4 and coreceptors (notshown). Formation of the prehairpin intermediate exposes the N-terminalcoiled coil (gray), the target of C-peptide inhibition. This transientstructure collapses into the trimer-of-hairpins state that brings themembranes into close apposition for fusion. FIG. 1B shows the design ofthe 5-Helix construct. The ribbon diagrams (top) depict the corestructure of the trimer-of-hairpins (left and D.C. Chan et al., Cell 89,263-273 (1997)) and a model of 5-Helix (right). The inner gray helicesrepresent N36 peptides, and the outer blue helices represent C34peptides. One C-peptide has been removed in the model of 5-Helix andorange lines have been drawn to represent connectivity between thehelices. In the design of 5-Helix, the N40 (SEQ ID NO: 3) and C38 (SEQID NO: 4) sequences (given in single-letter amino acid code) arealternately linked by short Gly/Ser peptide sequences (gray bars inschematic at bottom (See Example 1).

FIGS. 2A-2D show properties of 5-Helix. FIG. 2A is the circulardichroism (CD) apectrum of 5-Helix (10 μM) at 25° C. The spectrumindicates that the 5-Helix protein adopts >95% of the helical contentexpected from the design. FIG. 2B is a graphic representation of thermaldenaturation of 5-Helix monitored by ellipticity at 222 nm in TBS(filled squares) and in 3.7 M guanidine (Gu)HCl/TBS (open squares). Thedenaturation observed in the GuHCl solution is >90% reversible. FIG. 2Cshows results of nickel (Ni)-NTA precipitation of 5-Helix with aHis-tagged C-peptide. Untagged 5-Helix and His-tagged C-peptide (denotedC37-H6) were mixed before Ni-NTA agarose was added in order toprecipitate complexes containing C37-H6 (lanes 1 and 5 and Example 4).Addition of excess untagged C-peptide (C34) shifts the 5-Helix moleculesfrom the bound to the unbound fraction (lanes 2 and 6). FIG. 2D is theCD spectra of 5-Helix and C37-H6 before (filled squares) and after (opencircles) mixing in a mixing cuvette. The increase in ellipticity at 222nm upon mixing indicates an interaction between the two species thatincreases the total helical content (corresponding to an additional 28helical residues per associated C-peptide).

FIGS. 3A-3C show results of assessment of 5-Helix inhibition of HIV-1envelope-mediated membrane fusion, as described in Example 2. FIG. 3Ashows results of assessment of titration of viral infectivity by 5-Helix(filled squares), 6-Helix (open triangles), and 5-Helix(D4) (opencircles), as described in Example 3 and 5 The data represent themean±SEM of two or more separate experiments. FIG. 3B is a graphicrepresentation of antagonistic inhibitory activities of 5-Helix and C34.The number of syncytia were measured in a cell-cell fusion assayperformed in the absence or presence of 5-Helix, C34, or mixtures of5-Helix and C34 at the indicated concentrations. The IC₅₀ values for5-Helix and C34 in this assay are 13±3 nM and 0.55±0.03 nM, respectively(D. C. Chan et al., Proc. Natl. Acad. Sci. USA 95, 15613-15617 (1998)).Data represent the mean and range of mean of duplicate measurements,except for the control (mean±SEM of five measurements). FIG. 3C showsresults of assessment of 5-Helix inhibition of pseudotyped viruscontaining different HIV-1 envelope glycoproteins. The reported IC₅₀values represent the mean±SEM of three independent experiments.

FIG. 4 is a helical wheel diagram depicting the interaction of 5-Helixwith the C-peptide region of gp41. The a through g positions in eachhelix represent sequential positions in the 4,3-hydrophobic heptadrepeat in each sequence. The a and d positions in the gp41 C-peptideregion interact with the exposed e and g positions on the N40 coiledcoil of 5-Helix. Residues are boxed according to their degree ofconservation as determined from the alignment of 247 sequences fromHIV-1, HIV-2, and SIV isolates (HIV-1 sequence database, August 2000,Los Alamos National Laboratory): black rectangle, >90% identical; greyrectangle, >90% conservative substitution; dotted rectangle, 70-90%conserved; no box, <70% conserved. In generating FIG. 4, substitutionswithin the following groups of amino acid residues were considered to beconservative: [Asp, Glu], [Lys, Arg], [Asn, Gln], [Phe, Tyr], [Ser, Thr]and [Val, Ile, Leu, Met]. Note the high degree of conservation in the aand d positions of the C-peptide region of gp41, a property markedlylacking in other positions (particularly c and g) of the C-peptideregion not directly involved in binding 5-Helix.

FIG. 5 is the structural arrangement of HIV gp41. Helical regions(heptad repeats) are shown in grey, and the relative position of N—(N36)and C—(C34, DP178) peptides are indicated. In the ribbon diagram of thehelical region, the N-peptides are in light grey, while the C-peptideare in dark grey.

FIG. 6 is the sequence of 6-Helix (SEQ ID NO: 2) and 5-Helix (SEQ ID NO:1). The predicted helical segments are designated by the stackedsequence.

FIG. 7 is a ribbon diagram of one of the possible α-helical arrangementsof 5-Helix. The N-helical trimer is light grey, the C-helical regionsare in dark grey and the extended loop regions are in black (based onthe structure of D. C. Chan, et al. (Cell 89, 263-273 (1997)).

FIG. 8 shows images of cell-cell fusion assay titration experiments. Thesyncytia (representing fused cells) are blue in the image while debrisis brown.

FIG. 9 shows images of cell-cell fusion assay competition experiments.The amount of syncytia are recorded for cultures incubated in 200 nM6-Helix or 5-Helix with increasing amounts of C34 peptide.

FIG. 10 is a schematic of the design of the Five-Helix constructs. Theschematic diagram depicts the linkage pattern of the basic 5-Helixconstruct. Three different C-termini were added. In 6-Helix, aHis-tagged C-peptide is attached to 5-Helix in order to mimic thecomplete six-helix bundle of the trimer-of hairpins. The N40 and C38sequences (alternately joined using short Gly/Ser linkers) are derivedfrom the N- and C-peptide regions of HIV HXB2 gp41. (The red- andblue-boxed residues depict the sequences of the N36 (SEQ ID NO.: 11) andC34 (SEQ ID NO.: 12) peptides, respectively.)

FIGS. 11A-11C show amino acid sequences of peptides (SEQ ID NOS.: 1-10)of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The conformation of a major part of the ectodomain of the gp41 moleculeconsists of a trimer-of-hairpins structure. The core“trimer-of-hairpins” is comprised of a central three-stranded N-helixcoiled coil surrounded by three outer C-helices, forming a bundle with atotal of six helices. The trimer-of-hairpins is a common structuralelement involved in the fusion of many enveloped viruses, suggesting acritical role for this motif in promoting membrane fusion. In HIV gp41,the core of the trimer-of-hairpins is a bundle of six α-helices (formedby the C-terminal regions of three gp41 ectodomains) packed in anantiparallel manner against a central, three-stranded coiled coil(formed by the N-terminal regions of the gp41 molecules) (M. Lu et al.,J. Mol. Biol. 290, 1031-1044 (1995); D. C. Chan et al., Cell 89, 263-273(1997); W. Weissenhorn et al., Nature 387, 426-430 (1997)); K. Tan etal., Proc. Natl. Acad. Sci. USA 94, 12303-12308 (1997). Because thefusion peptide region, which inserts into the cellular membrane, islocated at the extreme N-terminus of gp41, and the C-terminal region isadjacent to the transmembrane helix anchored in the viral membrane, thetrimer-of-hairpins motif serves to bring the two membranes together.This is illustrated schematically in FIG. 1A. The N-helices (one fromeach subunit of the trimer) form highly conserved hydrophobic groovesinto which the C-helices pack. It is generally agreed that formation ofthis six-helix structure is required for membrane-fusion to occur.

The importance of trimer-of-hairpins formation for HIV-1 entry led tothe hypothesis that the C-terminal region of gp41 might serve as atarget for potential membrane-fusion inhibitors. C-peptides have beenshown to inhibit HIV-1 entry into cells, with IC₅₀ values as low as 1 nMin vitro (C. T. Wild et al., Proc. Natl. Acad. Sci. USA 91, 9770-9774(1994); D. C. Chan et al., Proc. Natl. Acad. Sci. USA 95, 15613-15617(1998)). Evidence suggests that C-peptides work in a dominant-negativefashion by binding to the N-peptide region and disruptingtrimer-of-hairpins formation. If the C-terminal region is accessible (atleast transiently) prior to formation of the trimer-of-hairpins, then itis reasonable to expect that agents that bind to this region of gp41N-terminal will prevent membrane fusion. Consistent with this notion,peptides derived from the gp41 N-terminal region (referred to asN-peptides) are modest inhibitors of HIV-1 membrane fusion. Theinhibitory mechanism of N-peptides has not been determined, in partbecause these peptides have a strong tendency to aggregate.

Applicants reasoned that a single soluble molecule that contains afolded N-helical core and two of the three C-helices of the coretrimer-of-hairpins would be highly stable and would bind a singleC-peptide with high affinity. As described herein, the hypothesis thatthe C-peptide region of gp41 is a target for inhibition of HIV-1 entryhas been tested. Results of the assessment, also described herein, haveshown that Five-Helix, which binds the C-peptide region of gp41, showspotent inhibitory activity against HIV-1 and against HIV-1 variantscontaining a diverse set of envelope proteins. These results point tothe C-peptide region of HIV gp41 as a viable target to inhibit theformation of the trimer-of-hairpins, which is required for membranefusion (and, thus, HIV infection of cells) to occur.

Described herein are results that show that a protein that binds to theC-peptide region of gp41 inhibits HIV entry into cells. Such proteinsare inhibitors of HIV and serve as the basis for development ofadditional anti-HIV agents. They might also be used for generating aneutralizing antibody response that targets the N-terminal region of thegp41 ectodomain.

Five-Helix, as the proteins are designated, takes advantage of thebinding properties of the N-helix peptide coiled coil while minimizingthe tendency of the N-peptides to aggregate. In one embodiment ofFive-Helix, five of the six helices that make up the core of the gp41trimer-of-hairpins structure are connected with (joined by) shortpeptide linkers. (See FIG. 1A.) In this embodiment, Five-Helix lacks athird C-peptide helix, thus creating a vacancy in order to create ahigh-affinity binding site for the C-terminal region of gp41. In furtherembodiments of Five-Helix, the three N-peptide helices and more than two(but less than three complete) C-peptide helices are connected withshort peptide linkers. In these embodiments, the three N-peptidehelices, two complete C-peptide helices and a portion of the thirdC-peptide helix are connected with peptide linkers. The portion of thethird C-helix can be as few as one amino acid residue of the thirdC-helix or any number of additional amino acid residues of the helix upto, but not including, all of the amino acid residues of the helix.Five-Helix protein of the present invention is soluble underphysiological conditions.

The core of the trimer-of-hairpins, as formed by individual N- andC-peptides, is already quite stable, with a melting temperature of 65°C. Applicants have shown that if 5 of the 6 helices are covalentlyjoined to form a 5-Helix protein, the stability of the core is furtherincreased (the stability is greater than the stability of the 6-Helixcore). Under physiological conditions, Five-Helix is folded, soluble,and stable. It has an α-helical content in close agreement with thevalue predicted from the design. (See FIGS. 2A and 2B.) Inaffinity-interaction experiments, Five-Helix interacts strongly andspecifically with epitope-tagged C-peptides. (See FIG. 2C.) Thisinteraction induces a helical conformation in the bound C-peptide asjudged by the difference in circular dichroism before and after mixing.(See FIG. 2D.) These properties are consistent with the intended designof Five-Helix.

Five-Helix potently inhibits HIV-1 membrane fusion (nanomolar IC₅₀) asmeasured by viral infectivity and cell-cell fusion assays. (See FIGS. 3Aand 3B.) In contrast, a control protein, denoted Six-Helix, in which theC-peptide binding site is occupied by an attached C-peptide (i.e., allsix helices that constitute the gp41 trimer-of-hairpins have been linkedinto a single polypeptide, as described in Example 1), does not haveappreciable inhibitory activity. (See FIG. 3A and FIGS. 8 and 9).Similarly, a Five-Helix variant, denoted Five-Helix(D4), in which theC-peptide binding site is disrupted by mutation of four interfaceresidues (V549, L556, Q563 and V570) to Asp, does not block the membranefusion event even at 1 μM. (See Example 3 and FIG. 3A.) These resultssupport the conclusion that C-peptide binding is the key determinant ofantiviral activity in Five-Helix.

The inhibitory activities of 5-Helix and C-peptides are expected to beantagonistic: when 5-Helix binds C-peptide, the amino acid residuesthought to be responsible for the antiviral activities of each inhibitorare buried in the binding interface. Indeed, mixtures of 5-Helix and C34[a well characterized and potent peptide inhibitor with an IC₅₀ ofapproximately 1 nM] display a dose-dependent antagonistic effect (FIG.3B). In the presence of 5-Helix, high-potency inhibition by C34 is onlyobserved when the peptide is in stoichiometric excess (FIG. 3B).

Five-Helix inhibits infection by viruses pseudotyped with a variety ofHIV-1 envelope proteins (from clades A, B, and D) with similar potency(FIG. 3D). This broad-spectrum inhibition likely reflects the highlyconserved interface between the N- and C-terminal regions within thegp41 trimer-of-hairpins structure (FIG. 4). The residues in theC-peptide region of gp41 that are expected to make contact with 5-Helixare highly conserved in HIV-1, HIV-2, and SIV (FIG. 4).

As a potent, broad-spectrum inhibitor of viral entry, Five-Helix mayserve as the basis for the development of a new class of therapeuticagents against HIV-1. Although they typically require parenteraladministration, protein-based therapeutics can be practical, asexemplified by insulin, growth hormone, tissue plasminogen activator,granulocyte-colony stimulating factor, and erythropoietin.Alternatively, Five-Helix could be expressed endogenously (e.g., viagene therapy) with secretion into the bloodstream. If Five-Helix wereexpressed endogenously in HIV-infected cells, it could inhibitintracellular folding and transport of gp160. Five-Helix, Five-Helix(D4), and Six-Helix are also potential reagents for small-moleculedrug-screening purposes. Five-Helix offers a great deal of flexibilityin the design of variants with better therapeutic characteristics. Inprinciple, Five-Helix can be modified extensively, except at itsC-peptide binding site, to alter its immunogenic, antigenic,bioavailability, or inhibitory properties. For example, the C-peptidebinding site might be lengthened, shortened, or shifted in the gp41sequence in order to optimize inhibitory potency by targeting differentregions of the gp41 ectodomain.

It would be desirable to generate neutralizing antibodies that mimic thebinding properties of Five-Helix. The broadly neutralizing ability ofFive-Helix most likely stems from its interaction with the highlyconserved residues in the C-peptide region of gp41 (FIG. 4).Unstructured C-peptide immunogens may not elicit broadly neutralizingantibodies because the linear sequence of the gp41 C-peptide region isvariable among different HIV-1 strains. Such unstructured C-peptides donot have a long region of conserved amino acids residues. Rather,conserved amino acid residues and nonconserved residues areinterspersed. However, constraining C-peptides or C-peptide analoguesinto a helical conformation (e.g., as in the C-peptide region when itbinds Five-Helix) may lead to useful immunogens in the effort to developan AIDS vaccine. FIG. 4 is a helical wheel diagram depicting theinteraction of Five-Helix with the C-peptide region of gp 41. As shown,on the helical wheel, the whole “face” is comprised of conserved oridentical amino acid residues. As also shown, there is a high degree ofconservation in the a and d positions of the C-peptide region of HIVgp41. Peptides from the C-terminal region of the gp41 ectodomainconstrained in such a manner that they present highly conserved aminoacid residues on a single face of the molecule (such as in positions a,d and e in FIG. 4) can be produced. They can be used as immunogens toproduce antibodies that will presumably bind those amino acid residuesin the corresponding unconstrained peptide (C-peptide region of HIVgp41) and, thus, mimic the binding characteristics of Five-Helix. Forexample, antibodies that bind some or all of the highly conserved(identical and/or conserved) amino acid residues in C38 (see FIG. 4) canbe produced. Such antibodies, which mimic the binding of Five-Helix,will work, in effect, as a preventive or vaccine by reducing orpreventing the activity (binding) of Five-Helix. Such antibodies toconstrained peptides from the C-terminal region of HIV gp41 ectodomainare a subject of this invention.

Intriguingly, the epitope for 2F5, the only known human monoclonalantibody directed against gp41 with broad neutralizing activity, islocated immediately C-terminal to the C-peptide region targeted byFive-Helix (T. Muster, et al., J. Virol. 67, 6642-6647 (1993); M.Purtscher, et al., AIDS 10, 587-593 (1996)). The core of the 2F5 epitope(Leu-Asp-Lys-Trp; residues 663-666 in the HIV HXB2 gp 160 sequence) ishighly conserved (81% identity) across the same set of HIV-1, HIV-2, andSIV isolates used to generate FIG. 4. However, some HIV-1 escapevariants to 2F5 neutralization do not contain mutations in the epitopesequence, suggesting that inhibition by 2F5 may involve recognition ofadditional determinants. The conformation of the 2F5-bound epitoperemains unknown, but antibodies elicited with fragments of gp41containing this sequence do not possess significant virus-neutralizingactivity (T. Muster, et al., J. Virol. 68, 4031-4034 (1994); L. Eckhart,et al., J. Gen. Virol. 77, 2001-2008 (1996)). It remains to be seen if2F5 inhibits infection by interfering with trimer-of-hairpins formation.

Further, Five-Helix itself is a vaccine candidate. The possibility ofeliciting an antibody response against transiently exposed conformationsof proteins involved in HIV-1 fusion has been suggested (R. A. LaCasse,et al., Science 283, 357-362 (1999)). One possible well-defined targetis the N-terminal coiled coil that is exposed in the prehairpinintermediate (D. M. Eckert, et al., Cell 99, 103-115 (1999)). A5-Helix-like intermediate may be exposed during the fusion process, and,in this case, antibodies directed against 5-Helix may inhibit viralentry.

Results described herein point to the C-peptide region of HIV-1 gp41 asa viable target to inhibit the formation of the trimer-of-hairpins.Structural and computational methods predict similar trimer-of-hairpinsmotifs for viruses in many diverse families, including orthomyxoviridae,paramyxoviridae, filoviridae, retroviridae, and others. Moreover, insome of these cases, inhibition of viral entry by peptides analogous tothe C-peptides of gp41 has been demonstrated. Thus, the Five-helixdesign approach may offer a widely applicable strategy for inhibitingviral infections.

In addition, Five-Helix provides a means to study a formed C-peptidebinding site in detail, which cannot be done with aggregable N-peptides.The exposed C-peptide binding site in this Five-Helix molecule is usefulto identify or design molecules that bind to the N-helical core of gp41and can be further assessed, using known methods, for their ability toinhibit fusion of the HIV membrane with the membrane of a mammaliancell, such as a human cell, thus inhibiting (reducing or preventing)infection of the cell. Further, Five-Helix can be assessed for itsability to bind to the C-helical region of gp41 and inhibit itsfunction. The N-helical core of gp41 is highly conserved (in terms ofamino acid composition) and, thus, it is likely that 5-Helix andvariants thereof will be broadly neutralizing against a variety ofclinical HIV strains and, thus, useful therapeutically.

The Five-Helix protein, which is based upon the known structure of thegp41 ectodomain, consists, in one embodiment, of three N-peptides andtwo C-peptides covalently linked and arranged to fold into a substantialpart of the N-helical core with two of the three C-helix binding sitesoccupied by C-peptide. The remaining C-peptide binding site of theN-peptide is exposed. The site exposes an epitope that is 40 amino acidsin length. In addition, it is expected that the backbone atoms of thesite are rigidly held in a structured conformation, as the N-peptidecore is locked into place by the outer two C-peptides.

In single letter amino acid code, the amino acid sequence of oneembodiment of Five-Helix is the following:

(SEQ ID NO.: 1) MQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGSSGGQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGSSGGQLLSGIVQQQNNLLRAIEAQQHL LQLTVWGIKQLQARILAGGR.

In single letter amino acid code, the amino acid sequence of 6-Helix isthe following:

(SEQ ID NO.: 2) MQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGSSGGQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGSSGGQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGRGGHTTWMEWDREINNYTSLIHSLIEESQNQ QEKNEQELLGGHHHHHH.

Five-Helix protein can be produced by a variety of methods. For example,it can be produced, as described in Example 1, from a larger protein,such as 6-Helix, by enzymatic (trypsin) digestion. Alternatively, it canbe produced, using known methods and expression systems, by expressingFive-Helix protein-encoding DNA, which can be a single DNA that encodesthe entire Five-Helix protein or two or more DNA “units”, each of whichencodes a portion (e.g., one or more N helices, one or more C helices)of N-Helix protein. The yield of expression and purification ofFive-Helix can be significantly improved by direct expression of theFive-Helix gene in an appropriate host cell, such as E. coli. In thisapproach, the Five-Helix gene encodes the residues present in the finalFive-Helix protein; A C-terminal His-tag can be attached to facilitatepurification (with or without a protease cleavage site to later removethe tag). The protein can then be used directly without the proteolyticcleavage and unfolding steps required for producing Five-Helix startingfrom Six-Helix. This Five-Helix molecule may be expressed as a foldedactive molecule, allowing its use in biological selections or screensfor optimizing its properties. Alternatively, protein synthetic methodscan be used to produce Five-Helix protein. The five helices ofFive-Helix can be joined covalently (such as by means of a linker of atleast one (one or more) amino acid residues) or by other means whichresults in formation of a protein which is stable under physiologicalconditions and is correctly folded such that the remaining surface ofFive Helix is presented so that it is available to bind C34 peptide. Inthe embodiments in which there are three N-helices and more than two(but less than three complete) C-helices, the helices can be similarlyjoined.

Five-Helix can have a wide variety of sequences, both in the N- andC-helix regions and in the linker components, and can be comprised ofL-amino acid residues, D-amino acid residues or a combination of both L-and D-amino acid residues. The amino acids residues can be modified.Five-Helix can include amino acid residues in addition to those of thehelices and linkers (e.g., to stabilize the molecule). It is likely thatthe Five-Helix described here can be altered to enhance stability andactivity. Minor changes in the design of the loops connecting the N- andC-helices (both in length and composition) and the exact borders of theN- and C-helices are likely to have significant effects on thestability, yield, and activity of Five-Helix.

As currently constructed, Five-Helix exposes a C-peptide binding siteencompassing 40 amino acids along the N-helical core. A strategy forexposing shorter segments of the C-peptide binding site on 5-helix (orrelated molecules) involves attaching a short C-peptide sequence ontothe longer exposed epitope. A molecule of this type might aid in thedevelopment of drugs targeted specifically to a shorter epitope alongthe N-helical core. For instance, a single pocket region (similar tothat found in IQN17; D. M. Eckert, et al., Cell 99, 103-115 (1999))could be exposed in Five-Helix by binding a C-peptide that lacks theresidues that bind there (the first 10 or so residues of C34). Theseshort C-peptide sequences could be attached to Five-Helix through avariety of means, including covalent crosslinking or merely extendingthe sequence of Five-Helix to cover part of the exposed epitope.

Five-Helix is useful in a variety of contexts. As described herein,Five-Helix is a potent inhibitor of viral membrane fusion, and, thus,acts on the virus before it enters the cell (unlike current practicaltherapy) which acts on HIV-infected cells. Five-Helix is soluble and hasbeen shown to be stable under the conditions described herein. It shouldalso be possible to generate 5-Helix variants with an increasedmolecular weight (by oligomerization or tethering to a large protein) toreduce the rate of kidney clearance. In addition, Five-Helix dimers canbe made by disulfide crosslinking, to produce a molecule filtered to alesser extent than the Five-Helix “monomer”. Thus, it is reasonable toexpect that dimers might have an enhanced bioavailability when comparedto that of the C-peptides.

Five-Helix prevents virus from entering cells, unlike standard therapythat targets viral proteins after viral entry, and thus, Five-Helix canbe used prophylactically to prevent infection or reduce the extent towhich infection occurs. One use for such a therapeutic is in the eventof a needlestick injury, such as might occur in a hospital or insettings in which needles contaminated with HIV are shared. For example,an individual who is stuck with a needle and is or might be infectedwith HIV can receive a sufficient quantity of Five-Helix(therapeutically effective quantity) in one or more dose(s) in order toprevent or reduce HIV entry into cells. Five-Helix can be administered,for example, by intravenous or intramuscular injection.

In one embodiment of the present invention, Five-Helix is used to reduceHIV infection in an individual. In this embodiment, Five-Helix isadministered, either as Five-Helix itself or via expression ofFive-Helix-encoding DNA in appropriate host cells or vectors, to anindividual in sufficient quantity to reduce (totally or partially) HIVinfection of the individual's cells. That is, a dose of Five-Helixsufficient to reduce HIV infection (an effective dose) is administeredin such a manner (e.g., by injection, topical administration,intravenous route) that it inhibits (totally or partially) HIV entryinto cells. In one embodiment, a gene therapy approach is used toprovide the effective dose, by introducing cells that express Five-Helixprotein into an individual. Five-Helix can be administered to anindividual who is HIV infected to reduce further infection, or to anuninfected individual to prevent infection or reduce the extent to whichinfection occurs.

Pharmaceutical compositions which comprise Five-Helix in an appropriatecarrier (e.g., a physiologically acceptable buffer) are a subject ofthis invention. They are useful for preventive and therapeutic purposesand can be administered via a variety of routes (e.g., injection,topical administration, intravenous route).

Five-Helix appears to present a single, intact C-helix binding site and,thus, is useful for screening for drugs that inhibit membrane fusion.Five-Helix exposes a larger, more rigid target for potential drugscreens than does IQN17. The molecules 6-Helix and 5-Helix(D4) are auseful negative control in these studies.

The Five-Helix exposed epitope can also be used as an antigen forproducing antibodies, particularly neutralizing antibodies using knownmethods. The antibodies can be monoclonal or polyclonal.

The serum stability of Five-Helix can be tested, using known methods, toascertain its therapeutic potential. If Five-Helix is degraded, the mostlikely point of attack/degradation is the glycine/serine linker regions.In this case, different linker regions can be generated and tested (seebelow). The inhibitory ability of these anti-Five-Helix sera and ascitescan be tested using standard fusion assays.

The outside surface of Five-Helix can be varied, for example, to enhancebioavailability, decrease toxicity and avoid immune clearance. SinceFive-Helix exhibits potent inhibitory activity, whereas the 6-Helixbundle does not, it is the exposed groove, including the pocket region,that is responsible for inhibition. The rest of the molecule simplyprovides a scaffold for displaying the exposed groove. Therefore, thisscaffold can be modified without adversely affecting the inhibitoryactivity of Five-Helix. Modification of the scaffold may provide severaladvantages. First, it would facilitate procedures in which multipleadministrations of Five-Helix are required. For example, when Five-Helixis used as an anti-HIV therapeutic agent, multiple doses might berequired. After extended administration, individuals might developantibodies to Five-Helix that are likely to increase its clearance fromthe body. The availability of multiple versions of 5-Helix would help tocircumvent this problem by evading pre-existing antibodies. Second, itmay be possible to design versions of Five-Helix, for example byintroducing glycosylation sites on the external surface, in which thescaffold is less immunogenic. For vaccine studies, this modificationwould help to bias the immune response toward the exposed groove asopposed to the scaffold.

The observation that binding the gp41 C-helical region prevents HIVinfection suggests a strategy for constructing an HIV vaccine. Analogousto inhibition of HIV by C-peptides, Five-Helix likely inhibits gp41 bybinding to a fusion intermediate of gp41 called the prehairpinintermediate. Whereas the C-peptide inhibitors function by binding tothe N-peptide region of this intermediate, Five-Helix likely functionsby binding to the C-peptide region. These considerations stronglysuggest that the C-peptide region of gp41 is a good drug target for thedevelopment of HIV entry inhibitors. Moreover, it may be possible to useC-peptide-based constructs as immunogens to elicit neutralizingantibodies. In the case of Five-Helix, the target of inhibition is ahelical conformation of the C-peptide region, but reagents targetingother conformations of the C-peptide region may also have inhibitoryactivity.

Recent vaccine studies (R. A. LaCasse, et al., Science 283, 357-362(1999)) suggest that intermediates of the envelope-mediated fusionprocess can elicit strongly neutralizing antibodies. Antibodies to suchfusion intermediates would target conserved regions of the envelopeproteins and therefore would be likely to neutralize a broad range ofviral strains. Antibodies to the C-peptide region would target a regionthat is highly conserved and critical to the fusion process.

The trimer-of-hairpins is a common feature of many viral membrane fusionproteins. It has been observed in crystal structures of Influenza,Ebola, SV5 (simian parainfluenza virus 5), and RSV (human respiratorysyncitial virus). In addition, many other members of the retrovirus,paramyxovirus, and filovirus families are predicted to contain thismotif A similar structure has been observed in the associated vertebratevesicle fusion proteins. The basic strategy described herein can beapplied to any of these systems in order to inhibit fusion. One subjectof this invention is a method of inhibiting formation of thetrimer-of-hairpins of an enveloped virus (a virus that comprises a viralenvelope protein) by contacting the virus with a drug that binds a viralenvelope protein (e.g., the C peptide region of a viral envelopeprotein) and inhibits formation of the trimer-of-hairpins of theenveloped protein.

The present invention is illustrated by the following examples, whichare not intended to be limiting in any way.

Example 1 Production of 5-Helix

The design of 5-Helix was based on the N36/C34 six-helix bundle crystalstructure (D. C. Chan, et al., Cell 89, 263-273 (1997)). For the 5-Helixprotein, each peptide region was extended (compared with N36 and C34) bythree residues on its N-terminus and one residue on its C-terminus,generating the final N40 and C38 segments (representing residues 543-582and 625-662 of HIV-1 HXB2 gp160, respectively). Three N40 and two C38segments were joined using a -GGSGG- linker after N40 and a -GSSGG-linker after C38. All constructs include an N-terminal Met fortranslation initiation. Two distinct 5-Helix proteins that differ onlyat their C-termini were generated for this study: (i) His-tagged5-Helix, which ends in GG(H)6, and (ii) untagged 5-Helix, which ends in-GGR. In addition, a third construct, denoted 6-Helix, was generated inwhich the 5-Helix backbone was connected to the His-tagged C-peptide,C37-H6 (see Example 4), through a trypsin-cleavable linker (-GGR-) (seeFIGS. 10 and 11A-11C).

All DNA constructs were assembled from PCR cassettes sequentially clonedinto the pAED4 vector [D. S. Doering, P. Matsudaira, Biochemistry 35,12677-12685 (1996)] using E. coli XL1-Blue (recA-strain, Stratagene).All proteins were recombinantly expressed in E. coli strain RP3098 grownin 2xYT to an OD (590 nm) between 0.5-0.7 before induction with IPTG(0.4 mM) for 3 hours. Bacterial pellets were resuspended in Tris/NaClbuffers (Qiaexpressionist booklet, March 1999, Qiagen) supplemented withComplete EDTA-free protease inhibitor tablets (Roche), and subsequentlyfrozen at 20° C. until the day of purification. Thawed resuspensionswere lysed (sonication or French press) and centrifuged (35,000×g for 30minutes) to separate the soluble fraction from inclusion bodies.

His-tagged 5-Helix (generated from plasmid p-5HelixH6) was purifieddirectly from the inclusion bodies resuspended in 8 M urea in TBS (50 mMTris, pH 8.0, 100 mM NaCl) and 10 mM imidazole. The mixture wasclarified by centrifugation (35,000×g for 30 minutes) before binding toa Ni-NTA agarose (Qiagen) column at room temperature. Protein was elutedin 6 M urea/TBS/100 mM imidazole in 40 ml (−5 column volumes). Theprotein was refolded by slow dripping into a one liter, stirred solutionof 20 mM Tris (pH 8.0) at room temperature. Refolded protein was thenreconcentrated by passage over a Ni-NTA agarose column and eluted with20 ml (−2 column volumes) of 100 mM imidazole in TBS.

Untagged 5-Helix was produced via proteolysis of 6-Helix (see below) togenerate a 5-Helix/C37-H6 complex. Following digestion with trypsin(1:200 weight ratio in TBS at room temperature for 1 hour, Sigma), the5-Helix/C37-H6 complex was bound to Ni-NTA agarose and washedextensively to remove excess trypsin. The beads were resuspended in 8 MGuHCl/TBS and heated (70° C.) in order to denature the complex. Thenonbinding fraction, containing denatured 5-Helix, was sequentiallydialyzed into 8 M urea/20 mM Tris, pH 8.0 (4 hours at room temperature)and 4 M urea/20 mM Tris, pH 8.0 (overnight at 4° C.). The protein wasloaded onto a DEAE column (Fastflow, Pharmacia) and a reverse ureagradient (4 M to 0 M urea in 20 mM Tris, pH 8.0) was run over 20 columnvolumes in 4 hours at room temperature. The protein was eluted from theDEAE resin using a NaCl gradient (0 to 300 mM) in 20 mM Tris, pH 8.0 (10column volumes).

6-Helix (generated from plasmid p-6Helix) was purified directly from thesoluble fraction of the bacterial lysate. The solution was passed overNi-NTA agarose column and eluted with an imidazole gradient (10-250 mM)in TBS over 10 column volumes.

For all proteins, monomers were separated from aggregates by gelfiltration (Sephacryl S200 HR or Superdex 75) in TBS. The proteinswere >95% pure as judged by SDS-PAGE and can be concentrated to at least3 mg/ml. The concentrations of all peptides and proteins were determinedby absorbance at 280 nm in 6 M GuHC1 [H. Edelhoch, Biochemistry 6,1948-1954 (1967)].

Example 2 Assessment of the Specificity of 5-Helix/C-peptide Interactionand of Inhibition by 5-Helix of Membrane Fusion

The specificity of 5-Helix/C-peptide interaction has been tested using aHis-tagged C-peptide (C37-H6, independently expressed in E. coli andpurified through reverse-phase HPLC) and Ni-agarose precipitation. InTBS with 30 μM of C37-H6 , 16 μM of 5-Helix is completely precipitatedby Ni-agarose. Addition of 150 μM C34 (no His-tag, chemicallysynthesized and purified over HPLC) substantially reduces the amount ofprecipitated 5-Helix. The effective competition of C37-H6 and C34indicates that 5-Helix binds C-peptide in a specific manner. The CDexperiments and competitive binding assays suggest that 5-Helix foldsinto the predicted conformation. That is, the results support theprediction that 5-Helix contains an exposed C-peptide binding site.

Assays were carried out to assess the ability of 5-Helix to interactwith the C-region of gp41 and inhibit function of the fusion protein.This inhibition of membrane fusion by 5-Helix and 6-Helix was assessedusing a cell-based assay. Proteins 5-Helix and 6-Helix are seriallydiluted in modified DMEM media with 5% FCS and aliquoted into slidechambers. HELA cells (4×10⁴) expressing CD4 and coreceptors andcontaining a β-galactosidase gene under the control of the Tat promoterare added. CHO cells (2×10⁴) expressing gp160 (precursor protein togp120/gp41) and Tat are also added. The 400 μl miniculture is incubatedat 37° C. for 8 to 24 hours; fused cells (syncytia) will transcribe andtranslate β-galactosidase. The cells are fixed in gluteraldehyde andexposed to X-gal/Fe solution for one hour. Syncytia that containβ-galactosidase turn blue-green. In this assay, 5-Helix demonstrates apotent inhibition of syncytia formation, with an IC₅₀ of 10-20 nM; inone assay the IC₅₀ was 13 nM. 6-Helix does not block fusion appreciablyeven at 1 μM concentrations.

In order to verify the specificity of the 5-Helix exposed epitope as theinhibitory agent and to rule out a contaminant, mixing experiments withC-peptide have been performed. 5-Helix, at 200 nM concentration, ismixed with C34 at 100, 166, 190 and 210 nM. At the concentrations used,free 5-Helix and free C34 should inhibit almost all of the syncytia inthe miniculture. In 5-Helix/C34 mixes where C34 is in excess of the5-Helix (i.e., at 210 nM) syncytia formation is blocked, whereassyncytia formation is partially blocked in the 5-Helix/C34 mixes whereC34 concentration is less than that of 5-Helix (FIG. 3B). By contrast,C34 in the presence of 6-Helix blocks all syncytia formation.

The inhibitory potentials of 5-Helix and 6-Helix have been reproduced inviral fusion experiments. HIV, modified to contain a luciferase reportergene, is mixed with human osteosarcoma (HOS) cells expressing CD4 andcoreceptor in the presence of diluted protein for 6 hours at 37° C. Thevirus solution is replaced, and the HOS culture is incubated 48 hoursmore in fresh media. Luciferase activity is measured in a luminometer.In this assay, 5-Helix inhibits luciferase activity with an IC₅₀ lessthan 10 nM. Again, 6-Helix shows no appreciable block up to 1 μM (FIGS.3A and 3C).

Example 3 Design and Assessment of 5-Helix (D4)

In 5-Helix(D4), four highly conserved residues in the C-peptide bindingsite of His-tagged 5-Helix (Va1549, Leu556, Gln563, and Va1570) weremutated to Asp in the final (third) N40 segment. The construct[p-SHelix(D4)] was recombinantly expressed and purified in the samemanner as the His-tagged 5-Helix. The His-tagged 5-Helix and 5-Helix(D4)proteins have the same ellipticity: for both, [θ]₂₂₂=−28,100±1500 degcm² dmol⁻¹ (˜100% of the predicted helical content) at 4° C. in TBS, andboth proteins are extremely stable to thermal denaturation (Tm>98° C.)in TBS, as well as to GuHCl chemical denaturation (Cm values ˜6 M for5-Helix(D4); ˜7.2 M for the His-tagged 5-Helix) at 25° C. The slightlydecreased stability of 5-Helix(D4) likely reflects the low helicalpropensity and charge of the Asp residues which, in this context, areplaced within a predominantly hydrophobic groove on the surface of5-Helix.

Example 4 His-tagged C-peptide C37-H6

Peptide C37-H6 is a His-tagged C-peptide of the following sequence:GGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLGHHHHHH (SEQ ID NO.: 5). Thepeptide is derived from HIV-1 HXB2 residues 625-661 (underlined) andcontains the entire C34 sequence (W628 to L661). C37-H6 is produced fromthe tryptic digestion of a recombinantly expressed construct, p4-NC1.1,consisting of one N40 segment joined to C37-H6 through a -GGR-linker.Following expression, NC1.1 is purified from the soluble fraction ofbacterial lysates in the same manner as 6-Helix. Trypsin digestion (sameconditions as for untagged 5-Helix) generates C37-H6, which is thenpurified to homogeneity by reverse phase HPLC using a Vydac C-18 columnand a linear gradient of acetonitrile in water containing 0.1%trifluoroacetic acid. The identity of C37-H6 was confirmed by massspectrometry (MALDI-TOF, PerSeptive). Like C34, C37-H6 is a potentinhibitor of HIV-1 membrane fusion, with an IC₅₀≈1 nM in the cell-cellfusion assay.

The data in FIGS. 2A-2D were generated using the untagged version of5-Helix, but similar results were obtained with the His-tagged version[see Example 3]. The CD (Aviv 62 DS) experiments were performed in TBSbuffer unless otherwise stated. In FIG. 2B, the protein concentrationwas 1 mM for the TBS sample and 0.54 mM for the GuHCl/TBS sample. InFIG. 2D, a quartz mixing cell (Helma) with 1 ml chambers (4.375mm/chamber pathlength) was utilized. The polypeptides were at aconcentration of 5.9 mM (5-Helix) and 6 mM (C37-H6) in 20 mM Tris, pH8.0/250 mM NaCl before mixing.

The 5-Helix precipitation experiment (FIG. 2C) was performed in 20 mlTBS with 16 mM untagged 5-Helix, 30 mM His-tagged C37-H6, and/or 150 mMC34. The solution was added to 10 ml of Ni-NTA agarose and incubated atroom temperature for 10 minutes. After the unbound supernatant wasremoved, the beads were washed twice with 1 ml TBS and then eluted with500 mM imidazole. The Ni-bound and unbound samples were run on a 16.5%Tris-Tricine polyacrylamide gel (Biorad) and stained with Gel-code Blue(Pierce).

Example 5 His-tagged 5-Helix

All data in FIGS. 3A-3C were generated using His-tagged 5-Helix (seeExample 1). The cell-cell fusion assays (FIG. 3B) were performed asdescribed (D. C. Chan et al., Proc. Natl. Acad. Sci. USA 95, 15613-15617(1998)). Inhibition of viral infectivity was studied using a recombinantluciferase reporter assay slightly modified from that previouslydetailed (D. C. Chan, et al., Proc. Natl. Acad. Sci., USA, 95,15613-15617 (1998)). Briefly, pseudotyped viruses were generated from293T cells cotransfected with an envelope-deficient HIV-1 genomeNL43LucR⁻E⁻[B. K. Chen, et al., J. Virol. 68, 654-660 (1994)] and one offour gp160 expression vectors: pCMV-HXB2 (D. C. Chan, et al., Proc.Natl. Acad. Sci., USA, 95, 15613-15617 (1998), pEBB-JRFL (kindlyprovided by B. K. Chen), pSVIII-UG024.2, and pSVIII-RW020.5. Theplasmids pSVIII-UG024.2 and pSVIII-RW020.5 were obtained from the NIHAIDS Reagent Program (F. Gao, B. Hahn, and the DAIDS, NIAID) and codefor envelope protein from primary HIV-1 isolates. Supernatantscontaining virus were prepared as previously described (D. C. Chan etal., Proc. Natl. Acad. Sci. USA 95, 15613-15617 (1998)) and used toinfect either HOS-CD4 cells (HXB2 and UG024.2) or HOS-CD4-CCR5 cells(JRFL and RW020.5). Cells were obtained from the NIH AIDS ReagentProgram (N. Landau). In FIG. 3A, viral infectivity assays were performedin the standard 24-well format (D. C. Chan et al., Proc. Natl. Acad.Sci. USA 95, 15613-15617 (1998)). The data in FIG. 3C were obtained fromassays conducted in 96-well format: virus-containing supernatant (10 ml)and media (90 ml) were overlaid onto HOS cells at 50% confluency.Following two days of incubation at 37° C., the cells were harvested in100 ml lysis buffer (Luciferase Assay System, Promega), of which 10 mlwas analyzed per manufacturer's protocol. The IC₅₀ values werecalculated by fitting the 5-Helix titration data to a Langmuir function[normalized luciferase activity=1/(1+[5-Helix]/IC₅₀)].

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of eliciting an immune response to HIV in an individual,comprising introducing, by an appropriate route, a compositioncomprising a Five-Helix protein and a physiologically acceptablecarrier, wherein the Five-Helix protein is soluble under physiologicalconditions and comprises the three-N-helices and at least two, but notthree complete, C-helices of the trimer of hairpin structure of HIVgp41, wherein the component helices are separated by linkers, in a dosesufficient to elicit the immune response in the individual.
 2. Themethod of claim 1, wherein said Five-Helix protein comprises SEQ IDNO:
 1. 3. The method of claim 1, wherein said Five-Helix proteincomprises SEQ ID NO:
 7. 4. The method of claim 1, wherein saidFive-Helix protein comprises SEQ ID NO: 9.